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Background There is no consensus regarding large-scale use of benznidazole to treat Chagas disease, because of its toxicity and low efficacy during the chronic phase. Objectives To evaluate heart rate variability in chronic Chagas patients before and after treatment with benznidazole. Methods Twenty-one Chagas patients with positive blood cultures and/or PCR received benznidazole (5 mg/kg twice daily for 60 days) and were matched for age and gender with 24 Chagas individuals with negative blood cultures, as controls. R–R intervals were assessed in time and frequency domains using an autoregressive algorithm under three different conditions: baseline, cold face and active tilt tests. Power spectral densities in low and high-frequency bands were estimated in absolute and normalized units. Data were expressed as mean ± SD or medians (lower and upper quartiles). Groups were compared using non-paired or paired Student's T, Mann–Whitney or Wilcoxon tests, as required. The significance level was 5%. Results The groups were comparable regarding age, gender and clinical disease forms. There were no differences in temporal and spectral indices between groups in baseline and cold face tests. Treated patients presented significantly lower tachycardic responses in mean R–R intervals during the active tilt test (p = 0.0200) than controls did. Genetic characterization of T. cruzi isolates from treated group samples showed that 100% belonged to genotype II. Conclusion For the first time in the medical literature, we detected sympathetic impairment during the active tilt test in chronic Chagas patients treated with benznidazole. This finding may be partially explained by benznidazole neurotoxicity.